PROJECT SUMMARY/ABSTRACT Immune heterogeneity, mediated by epigenetic and genetic mechanisms, complicates our understanding of immune regulation and disease. As sentinels, immune cells constantly survey their environment, and this information is presumably encoded epigenetically to direct cellular responses. Despite this understanding, little is known about epigenetic variation in immune cells and how it relates to immune phenotype heterogeneity. It is essential to investigate the relationship between epigenetic variation and immune response heterogeneity in order to better understand individual immunity. We will study dendritic cells (DCs), cells of the innate immune system, that engage in distinct cellular programs in response to environmental stimuli and are essential for protective immune responses. OVERALL OBJECTIVES: Our objective is to determine whether chromatin states in DCs underlie immune phenotype variation. We will characterize the degree of diversity in the epigenome of five mouse strains with known immune phenotype variation. We will map chromatin accessibility (ATAC-Seq) and select histone PTMs using a novel low-input chromatin mapping technique Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and compare to gene expression in the steady state and following immune stimulation. We will determine the extent of gene or enhancer priming in DCs that occurs to enable rapid gene expression following stimulation and identify regulatory networks that drive DC function. Our studies will reveal the dynamic and static aspects of the epigenome in response to stimulation and determine the extent to which epigenetic programming underlies differential activation. We will begin to dissect the interplay between genetic and epigenetic variation in immune cells and how it relates to immune phenotype heterogeneity. These concepts have been poorly studied in in vivo-derived DCs due to technical limitations that have been overcome by recent advances in low-input technologies for genome-wide studies. We have assembled a team that will ensure the completion of this project. Our collaborators bring extensive expertise in bioinformatics and epigenetic techniques, and coupled with our extensive experience in DC biology, creates a unique team to address the proposed questions. IMPACT: Upon completion of this research, we will have provided a major conceptual advance defining the relationship between chromatin states and function in DCs. We will have described the static versus dynamic aspects of the epigenome in response to immune stimulation. These studies will also give us new insights into the importance of the chromatin architecture for immune phenotype variation. These studies will lay the foundation for future study of how environmental perturbation of the epigenome?by conditions such as inflammation, nutrition, obesity, and overall health?interacts with genetics to impact immune cell programming and immune response heterogeneity.